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Laboratory Diagnosis of Viral Infection

Olivia Wesula Lwande

Learning Objectives

Describe the principles, techniques, standards and recording of results and interpretation of different methods used in diagnosis of viral infections

Difficulties
Can not be seen under light microscope Can not be cultivated easily Do not grow on culture media Treatment was not available Changed situation Rapid techniques have emerged Screening for Blood transfusion Treatment available

Techniques used
Microscopy Detection of Viral Antigen Growing and detecting viruses in

Tissue / Organ / Cell culture Fertilized hens egg Laboratory animal inoculation eg mice

Detection of antobody in serum


IgG Rising titre in paired sample IgM Indicates current / recent infection

Microscopy
Electron Microscope / Immune Electron Microscopy Light microscope Inclusion bodies eg Negri Body in Rabies Fluorescent Microscope Fluorescent antibody technique

Precipitation

on gel eg HBsAg Immunofluorescence Counter Immuno Electro Phoresis (CIEP) Enzyme Linkes Immuno Sorbant Assay (ELISA)

Demonstration of Viral Antigens

Isolation of Virus
Laboratory animals Fertilized Hens Egg

Chorioallantoic membrane Allantoic cavity Amniotic cavity Yolk sac

Organ/Tissue/Cell Culture Growth identified by serological method like neutralization.

Virus Culture
Embryonated Egg
Chorioallantioc membrane (CAM) Allantoic cavity Amniotic cavity Yolk Sac

Cell Lines/ Tissue cultures

Primary Diploid/ Secondary Continuous

Animal inoculation

Suckling mice

Embryonated Hens Egg

Chorioallantoic membrane (CAM) visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus Allantoic cavity Influenza virus (vaccine production) & paramyxoviruses Amniotic cavity primary isolation of Influenza virus

Yolk sac Chlmyadia, Rickettsiae & some viruses

Embryonated Hens Egg

Inoculation

Harvesting

Cell Culture

Routinely used for growing viruses Classified into 3 types:


1. Rhesus monkey kidney 2. Chick embryo fibroblast 3. Human amnion cell culture

Primary cell culture normal cells freshly taken from body & cultured, limited growth

Diploid cell strains cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 50
1. WI-38: human embryonic lung cell 2. HL-8: Rhesus embryo cell

Continuous cell lines malignant cells, indefinite subcultivtion


1. 2. 3. 4. HeLa: Human Ca of cervix cell line HEP-2: Human epithelioma of larynx Vero: Vervet monkey kidney McCoy, Detroit-6, BHK-21, Kb

Cell Culture

Tissues
trypsin & mechanical shaking

Individual cells

Cells are washed, counted & suspended in a growth medium.


Growth medium Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator

Cell Culture Bottles / Tubes

Detection of virus growth in cell cultures


1.

Cytopathic effects (CPE) morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses Metabolic Inhibition no acid production in presence of virus Hemadsorption influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture

2.

3.

Detection of virus growth in cell cultures


4.

Interference growth of a non cytopathogenic virus can be tested by inoculating a known cytopathogenic virus: growth of first virus will inhibit the infection by second Transformation oncogenic viruses induce transformation & loss of contact inhibition microtumors Immunofluorescence test for viral Ag in cells from viral infected cultures.

5.

6.

Viral Hemagglutination

Hemagglutination
Originally seen with the Influenza virus by Hirst in 1941. A convenient method of detection & assay of Influenza virus. Due to the presence of Hemagglutinin spikes on the surface.

Viral Assay

Viral content of a specimen: Total no. of


1. Virus particles EM, HA 2. Infectious virions only

Assay of Infectivity: two types


1. Quantitative assays actual no. of infectious particle in an inoculum 2. Quantal assays indicate the presence or absence of infectious viruses, carried out in animals, eggs or tissue cultures

Viral Assay

Assay of Infectivity: Quantitative assays


Plaque assay in monolayer cell cultures Pock assay on CAM

*Each plaque/ pock represents one infectious virus. Plaques are clear zones that develop on lawns of host cells. The virus plaque is analogous to the bacterial colony.

Specimens
According

to the disease

Respiratory Throat swab CNS CSF Eyes- Conjunctival scrapings Liver Blood PUO Blood Skin - Scrapings

Serological Reactions

Rising titre of antibody in paired sample of sera is diagnostic


First sample At the earliest Second sample After 2 weeks

Single sample - IgM type of antibody detection. Indicates recent / current infection. Techniques Neutralization, ELISA, CFT, Haemagglutination Inhibition (HAI)Test